Spinal and bulbar muscular atrophy (SBMA) is a degenerative disorder of lower motor neurons and skeletal muscle caused by a CAG/glutamine tract expansion in the androgen receptor (AR) gene. The polyglutamine AR (polyQ AR) undergoes hormone-dependent nuclear translocation and unfolding, steps that are essential to toxicity and to the development of progressive muscle weakness in men. To alleviate this toxicity, we will pursue a novel strategy to degrade the disease-causing protein by targeting the Hsp90/Hsp70-based chaperone machinery. We have previously demonstrated that allosteric regulation of Hsp70 to favor the ADP- bound state promotes polyQ AR ubiquitination and degradation. The objective of this application is to identify a lead small molecule that stabilizes the ADP-bound state of Hsp70 and promotes clearance of the toxic protein. Our approach is based on preliminary studies demonstrating that the thermostability of Hsp70 increases by 13C when in the ADP-bound state. We initially used this assay to manually screen 727 small molecules from the NIH Clinical Collection library and found one compound, esomeprazole, that increases the melting temperature of Hsp70 by nearly 10C. We confirmed that activity of this compound is stereospecific, and that in secondary assays it enhances Hsp70-dependent ubiquitination and client protein clearance. As esomeprazole has low potency, we miniaturized this assay to 384-well format and performed a multiplexed, primary screen of 35,840 compounds (Z-score of 0.8). The assay identified 40 compounds with activity after deconvolution and dose titration. These initial results indicate that we have developed a robust high-throughput screen to search for a potent, more useful compound. We propose the following specific aims to identify a sufficiently active compound that will stabilize the ADP-bound conformation of Hsp70 and enhance ubiquitination and degradation of the polyQ AR. In the R21 phase of this application, we will complete a robust pilot screen for stabilizers of Hsp70 starting with a collection of 35,840 compounds. Additionally, we will develop and perform a ThermoFluor-based counter-screen and secondary screens to assess effects on ubiquitination and polyQ AR clearance using quantitative ELISAs. Quantitative milestones mark the R21/R33 transition. The R33 phase is aimed at performing the high-throughput screen of an additional 130,000 compounds, and optimizing actives by medicinal chemistry, NMR and computational approaches. At the completion of these studies, we expect to have identified one-two chemical scaffolds of small molecule stabilizers of Hsp70 that act to favor the ADP conformation and enhance ubiquitination and degradation of polyQ AR. Our long-term plans are to apply for a Blueprint Neurotherapeutics Network grant to facilitate development of the most promising agents to the clinic.